Method &amp; Medium for use in detecting E. coli and total coliforms

ABSTRACT

A selective culture medium which permits simultaneous detection of total coliform and Escherichia coli in a test sample with a single growth phase incubation period. The culture medium includes the required components of: (i) carbon nutrients, (ii) a nitrogen nutrient, (iii) a source of metabolizable potassium, (iv) a source of metabolizable phosphate, (v) vitamins, (vi) minerals, (vii) amino acids, (viii) sodium pyruvate, (ix) a bactericidal system selective for non-coliform bacteria, and (x) a sensible indicator selectively metabolized by Escherichia coli to the exclusion of other coliforms; and the optional components of: (xi) a source of metabolizable magnesium, (xii) sodium chloride as a recovery agent, (xii) a nonionic surfactant, (xiii) a contrast promoter effective for enhancing the color differentiation between Escherichia coli colonies growing in the medium and other bacterial colonies growing in the medium induced by the sensible indicator, and (xiv) a highlighting dye for enhancing the visual differentiation of coliform colonies relative to the culture medium.

This is a continuation of application Ser. No. 08/221,831, filed Apr. 1,1994, now abandoned.

FIELD OF THE INVENTION

The invention relates to the detection of microbes. More specifically,the invention relates to the detection of Escherichia coli and totalcoliforms.

BACKGROUND

Civilization has long recognized the relationship between fecalcontamination and the outbreak of disease. Two categories of bacteriaroutinely tested as indicative of fecal contamination are Escherichiacoli and total coliforms (typically Klebsiella sp., Citrobacter sp., andEnterobacter sp.). Because of the widespread testing for the presence ofthese two categories of microbial pathogens, substantial effort has beendevoted to developing detection systems which permit quick and accuratetesting of one or both of these bacterial groups.

Early methods for the detection of coliforms in food and water generallyincluded the steps of (i) inoculating a primary culturemedium--formulated to encourage growth of target pathogen(s) whilediscouraging the growth of others--with a specimen of the food/water tobe tested, (ii) incubating the inoculated medium under conditions whichpromote growth of the target pathogen(s), and then (iii) analyzing theincubated medium to identify and/or quantify the microbes grown on themedium. The specimen introduced into the culture medium may be anuntreated sample or a sample which has been treated to concentrate anytarget microbial pathogens present in the sample--such as by membranefiltration.

The three most common test methods for detecting coliforms are theMultiple Tube Fermentation technique (MTF), the Membrane Filtrationtechnique (MF) and the Presence-Absence technique (P/A). The MTFtechnique yields the Most Probable Number (MPN) while the P/A techniqueprovides a simple positive/negative answer. In contrast, the MFtechnique permits direct observation and counting of bacterial colonies.The MFT technique yields results in about twenty four hours (except forthe determination of fecal coliforms which typically requires seventytwo hours) but is difficult to interpret as sample turbidity increasesand is typically less precise than MF data.

Because of the difficulty in effectively controlling the growth of othermicrobial pathogens in culture media, it is frequently necessary toisolate a sample of the suspected target microbial pathogen from theincubated primary culture medium, inoculate a second culture medium withthe sample, incubate the second culture medium in order to obtain apurified sample of the microbe, and then test the purified sample toensure that the suspected target pathogen is--indeed--the targetpathogen. The need to conduct a second incubation greatly increases boththe time required to conduct the test and the cost of the test.

Various media have recently been developed which include a detectionsystem permitting differentiation of the target bacterial pathogen--suchas Escherichia coli and/or total coliforms--from other bacteria in theprimary medium. Such detection systems include a mechanism by which thetarget bacterial pathogen(s) is differentiated from other bacteria so asto prevent "false positive readings" while promoting sufficient growthand recovery of the target bacterial pathogen(s) so as to avoid "falsenegative readings". While simple in theory, formulation of culture mediahaving such dual function detection systems has proven difficult inpractice.

The industry has developed several acceptable test methods for detectingEscherichia coli and/or total coliforms but the search continues forsuperior culture media which can provide quick, accurate and easilyreadable results.

Examples of the various culture media which have been developed fordetecting coliform bacteria and/or Escherichia coli are disclosed inInternational Application WO 89/04372 issued to Berg, European PatentApplication Publication 0025467 issued to Rembach, U.S. Pat. No.4,925,789 issued to Edberg, U.S. Pat. No. 5,210,022 issued to Roth etal., and the journal article New Medium for the Simultaneous Detectionof Total Coliforms and Escherichia coli in Water by Brenner et al.published in Applied and Environmental Microbiology.

Berg (International Application WO 89/04372) discloses a culture mediumformulated to quickly detect the presence/absence of coliforms. Themedium includes lactose as a carbon nutrient, a methylumbilliferonesubstrate as a fluorescent indicator, and the anionic surface activeagent sodium lauryl sulfate as a means of accelerating hydrolysis of themethylumbilliferone by the coliforms present in the medium.

A commercially available culture medium--sold under the trademarkCOLIFAST™--is manufactured in accordance with the general preceptdisclosed in the Berg International Application. Briefly, COLIFAST™mandates a maximum eight hour incubation period and is capable ofdetecting either fecal coliforms or total coliforms but not both on thesame plate.

Rembach (European Patent Application Publication 0025467) discloses aculture medium formulated to detect the presence/absence of Escherichiacoli which includes the chromogenic substrate 8-hydroxyquinolineglucuronide in combination with X-glucuronide as an activator. Rembachindicates that the medium results in the selective formation of a blackprecipitate in colonies of Escherichia coli.

Edberg (U.S. Pat. No. 4,925,789) discloses a culture medium formulatedto detect the presence/absence of a target microbe by limiting thenutrients present in the medium to a nutrient which metabolizes to aunique detectible product (nutrient-indicator) and is capable of beingdetectably metabolized only by the target microbe. Edberg specificallydiscloses detection of Escherichia coli using a color-indicatingnutrient selected from o-nitrophenyl-β-D-glucuronide (yellow),p-nitrophenyl-β-D-glucopyranosiduronic acid (yellow),β-napthalamide-β-D-glucuronide (purple), α-naphthol-β-D-glucuronide(red) or methylumbilliferyl-β-D-glucuronide (fluorescent) as the primarynutrient in the medium. Edberg further suggests that the medium may beused to simultaneously test for Escherichia coli and total coliforms(EC/TC) by employing different color-indicating nutrients for each ofEscherichia coli and total coliforms in the medium.

A commercially available culture medium--sold by Environetics under thetrademark COLILERT™--is manufactured in accordance with the generalprecept disclosed in the Edburg '789 Patent. Briefly, the COLILERT™culture medium detects the presence/absence of Escherichia coli andtotal coliforms (EC/TC) by limiting the nutrients present in the mediumto o-nitrophenyl-β-D-galactopyranoside (yellow) which is metabolized bytotal coliforms and methylumbilliferyl-β-D-glucuronide (fluorescent)which is metabolized to a significant extent only by Escherichia coli.

Roth et al. (U.S. Pat. No. 5,210,022) discloses a culture mediumformulated to quantify the amount of both Escherichia coli and totalcoliforms in a test sample. The medium limits the primary carbonnutrient in the medium to a chromogenic β-galactosidase substrateproducing an insoluble precipitate of a first color when reacted upon byβ-galactosidase (produced by coliforms generally) and a chromogenicβ-glucuronidase substrate producing an insoluble precipitate of acontrasting color when reacted upon by β-glucuronidase (produced to asignificant extent only by Escherichia coli). Roth et al. discloses thatsince the chromogenic indicators are specific to coliforms, the medianeed not include any inhibitors. While generally effective as an EC/TCtesting medium, the medium of Roth requires an incubation period ofabout 24 to 48 hours and is difficult to read.

A commercial product designed to detect the presence of both Escherichiacoli and total coliforms in a test sample is sold by RCR Scientific,Inc. under the trademark COLICHROME 2. The system is based upon the dualchromogenic and/or fluorescent system disclosed by Roth et al. andincludes (i) a chromogenic β-galactosidase substrate which produces afirst color when hydrolyzed by β-galactosidase [produced by coliformsgenerally], and (ii) a chromogenic β-glucuronidase substrate(5-bromo-4-chloro-3-indoxyl-β-D-glucuronide acid cyclohexylammoniumsalt) which produces a second color when reacted upon by β-glucuronidase[produced to a significant extent only by Escherichia coli]. Whilegenerally effective as an EC/TC testing medium, the medium suffers fromthe same limitations as the medium disclosed by Roth et al.

Brenner et al., New Medium for the Simultaneous Detection of TotalColiforms and Escherichia coli in Water, Applied and EnvironmentalMicrobiology, 59:3535-3544 discloses a culture medium similar to thatdisclosed in Roth et al for quantify the amount of both Escherichia coliand total coliforms in a test sample. The medium includes a firstchromogenic substrate which produces a fluorescent detectable color whenreacted upon by β-galactosidase (produced by coliforms generally) and asecond chromogenic substrate producing a second color when reacted uponby β-glucuronidase (produced to a significant extent only by Escherichiacoli). While generally effective as an EC/TC testing medium, the mediumis difficult to read and includes anionic detergents which tend toarrest recovery of stressed coliforms.

As evidenced by the abbreviated discussion set forth herein, severalacceptable EC/TC detection systems are available. However, a substantialneed continues to exist for a quick, accurate and easily readableculture medium which effectively encourages the growth of totalcoliforms while inhibiting the growth of non-coliforms and provides anobservable differentiation between Escherichia coli and other coliformswithin the medium.

SUMMARY OF THE INVENTION

I have developed a selective culture medium which permits simultaneousdetection of total coliform and Escherichia coli in a test sample with asingle growth phase incubation period. The culture medium includes (i)carbon nutrients, (ii) nitrogen nutrients, (iii) a source ofmetabolizable potassium, (iv) a source of metabolizable phosphate, (v)vitamins, (vi) minerals, (vii) amino acids, (viii) sodium pyruvate, (ix)a bactericidal system selective against non-coliform bacteria, and (x) asensible indicator selectively metabolized by Escherichia coli to theexclusion of other coliforms.

The culture medium may optionally include (xi) a source of metabolizablemagnesium, (xii) sodium chloride as a recovery agent, (xiii) a nonionicsurfactant, (xiv) a contrast promoter effective for enhancing the colordifferentiation between Escherichia coli colonies growing in the mediumand other bacterial colonies growing in the medium induced by thesensible indicator, and (xv) a highlighting dye for enhancing the visualdifferentiation of coliform colonies relative to the culture medium.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING A BEST MODE

Definitions

As used herein, including the claims, the term "primary"--when used todescribe a component of the medium--means the predominant source forproviding the specified attribute, but not necessarily the only source.For example, lactose is the "primary" carbon source in the preferredembodiment of this invention even though several other components,including the casitone, yeast extract, and5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexane salts, alsoprovide metabolizable carbon.

As used herein, including the claims, the term "sodium pyruvate"includes both pyruvic acid (CH₃)COCOOH) and sodium pyruvate (CH₃)COCOO⁻Na⁺).

Composition of Medium

I have developed a selective culture medium which permits simultaneousdetection of total coliform and Escherichia coli in a test sample with asingle growth phase incubation period. The culture medium includes (i)carbon nutrients, (ii) nitrogen nutrients, (iii) a source ofmetabolizable potassium, (iv) a source of metabolizable phosphate, (v)vitamins, (vi) minerals, (vii) amino acids, (viii) sodium pyruvate, (ix)a bactericidal system selective for non-coliform bacteria, and (x) asensible indicator selectively metabolized by Escherichia coli to theexclusion of other coliforms.

The culture medium may optionally include (xi) a source of metabolizablemagnesium, (xii) sodium chloride as a recovery agent, (xiii) a nonionicsurfactant, (xiv) a contrast promoter effective for enhancing the colordifferentiation between Escherichia coli colonies growing in the mediumand other bacterial colonies growing in the medium induced by thesensible indicator, and (xv) a highlighting dye for enhancing the visualdifferentiation of coliform colonies relative to the culture medium.

The relative concentrations of components in the medium is selected toprovide a pH between 5.0 and 9.0 with a preferred pH of about 7±0.2.This pH favors the growth of coliform bacteria relative to non-coliformbacteria.

A variety of carbon nutrients for supporting coliform growth areavailable. The most commonly employed carbon nutrient in culture mediumsintended to support coliform bacteria is lactose. I have surprisinglydiscovered that lactose concentrations of greater than about 1 to 2 wt %do not significantly improve coliform growth in the culture and tend tointerfere with development of a distinctive red coloration in thecoliform colonies. Other components included in the culture medium mayalso provide carbon nutrients such as the peptones--which are added as aprimary source of metabolizable nitrogen.

Similarly, a variety of nitrogen sources suitable for use in the culturemedium of this invention are well known and widely available. A widelyused nitrogen source which is acceptable for use in the medium of thisinvention are the peptones, such as casitone. The presence of peptonesin the culture tends to aid in the recovery of stressed coliforms.Another suitable nitrogen source is ammonium sulfate.

Metabolizable potassium and phosphate sources are provided to promotebacterial growth. A number of commercially available compounds suitablefor use in the medium of this invention are available. A particularlysuitable material is potassium phosphate due to its ability to provideeffective amounts of both nutrients and buffer the medium against pHshifts caused by metabolism of the nutrients.

A source of vitamins, minerals, and amino acids is also included in themedium. While only small quantities of these compounds are required,they are essential for the coliforms to achieve maximal growth. Areadily available source for all of these essential compounds is thewidely available commercial preparation known as yeast extract. Yeastextract also contributes a metabolizable source of calcium which isbelieved to assist in the recovery of stressed coliforms.

Recovery of stressed coliforms is fostered by the inclusion of sodiumpyruvate, a well known bacterial protective agent capable of enhancingrecovery of coliform bacteria. When incorporated in the present medium,sodium pyruvate has been found to increase bacterial recovery of certainspecies from ten to one hundred fold. Recovery of stressed bacteria canbe further enhanced by the inclusion of a magnesium source, such asanhydrous magnesium sulfate, and a source of sodium, such as sodiumchloride, in the medium. Without intending to be limited thereby, it isbelieved that the presence of magnesium fosters the recovery of stressedcoliforms by increasing the strength of the membrane.

The growth of non-coliform bacteria is retarded by the inclusion of abactericidal system which includes one or more inhibitors which areselective for non-coliform bacteria. Particularly suitable bactericidalagents which are selective for a broad range of non-coliform bacteriainclude: dyes such as methylene blue (C₁₆ H₁₈ N₃ SCl·3H₂ O), and nileblue; antibiotics such as Cefsulodin™ (a third generation cephalosporinavailable from Sigma Chemicals), Erythromycin (selective against grampositive bacteria), monensin (selective against gram positive bacteria),and penicillin (selective against gram positive bacteria); and nonionicsurfactants, such as alcohol C₁₋₄ alkoxylates. The growth ofnon-coliform bacteria is also retarded by selection of an incubationtemperature which inhibits growth of the non-coliform bacteria. Inaddition, certain bacteria simply cannot effectively compete against thecoliform bacteria for nutrients and are "starved" by the coliformbacteria present in the medium.

A nonionic surfactant can be incorporated into the culture medium forpurposes of enhancing visability of the bacterial colonies by increasingcolony diameter. Inclusion of a nonionic surfactant also tends to reducefalse positive readings--particularly when testing natural watersamples--by inhibiting the growth of non-coliform bacteria.Substantially any of the well known nonionic surfactants may be usefullyemployed in the culture medium of this invention. A detailed list ofsurfactants is provided in McCutcheon's; Emulsifiers and Detergents,North American Edition, Volume 1, 1993. Specific nonionic surfactantssuitable for use in the culture medium include the C₁₋₄ alkoxylatedalcohols, such as the TRITON® X family of octylphenoxy polyethoxyethanol nonionic surfactants available from Union Carbide Chemical &Plastics Company. The type and amount of surfactant must be carefullyselected to limit the bactericidal effect of this component whilemaximizing the desired characteristics.

Detection of total bacterial colonies incubated on the medium isenhanced by the inclusion of a highlighting dye. The highlighting dyeprovides a visually perceptible color difference between the medium andthe bacterial colonies growing on the medium. Chromogenic substrateswhich produce a color change when metabolized by coliform bacteria areparticularly suitable for this purpose. Chromogenic substrates which aremetabolizable by non-coliform bacteria are also suitable as ahighlighting dye as the presence of non-coliform bacteria in the mediumis controlled by other means. One example of a suitable highlighting dyeis triphenyltetrazolium chloride (TTC) which is metabolizable bysubstantially all bacteria to produce a red pigment. The concentrationof TTC incorporated into the medium should be carefully monitored asexcessive TTC can inhibit the growth and recovery of coliforms.

A conventional culture medium gelling agent can optionally be includedin the medium of this invention as desired. While several solidifyingagents are recognized as acceptable for use in gelling culture mediums,agar is generally accepted as the industry standard. Alternatively, themedium can be absorbed in liquid form into sterile pads retained withinstandard MF-style petri plates.

The detection system of the medium is provided by the inclusion of asensible indicator which is selectively metabolized by Escherichia colito the exclusion of other coliforms. The selective nature of theindicator permits Escherichia coli colonies to be differentiated fromcolonies of other coliforms and thereby enables detection of bothEscherichia coli and total coliforms in the same plate. A group ofchromogenic substrates selective for Escherichia coli are the well knownβ-glucuronidase substrates. These substrates includes a chromogenicmoiety attached to a glucuronidase-metabolized moiety wherein the colorof the chromogenic moiety is concealed until the metabolizable moiety iscleaved by glucuronidase. Since Escherichia coli is the only coliformknown to express meaningful quantities of glucuronidase, a bacterialcolony in the medium tinted by the chromogenic reagent (based upon theknown metabolized color of the reagent) indicates the presence ofEscherichia coli, while those which are not tinted by the chromogenicreagent indicate the presence of another coliform species. Examples ofsuch selectively metabolizable compounds includes specifically, but notexclusively, orthonitrophenyl-β-D-glucuronide (yellow);β-napthalamide-β-D-glucuronide (purple); α-naphthol-β-D-glucuronide(red); methylumbilliferyl-β-D-glucuronide (fluorescent);5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt(blue); 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid sodium salt(blue), 5-bromo-4-chloro-3-indoxyl-β-D-glucuronide cyclohexylammoniumsalt (blue); 5-bromo-4-chloro-3-indoxyl-β-D-glucuronide sodium salt(blue); 4-methylumbelliferyl-β-D-glucuronide (fluorescent);6-chloroindolyl-β-D-glucuronide (mauve or magenta);4,6-dichloroindolyl-β-D-glucuronide (mauve or magenta);6,7-dichloroindolyl-β-D-glucuronide (mauve or magenta);4,6,7-trichloroindolyl-β-D-glucuronide (mauve or magenta); andindoxyl-β-D-glucuronide (blue). The preferred indicator--when the mediumis solidified by the inclusion of a solidifying agent or used inconjunction with an absorbent pad--is5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt(often referenced as X-Gluc or BCIG). X-Gluc forms an insolubleprecipitate when hydrolyzed by a β-glucuronidase which does not migratewithin the medium. This facilitates differentiation of Escherichia colicolonies from colonies of other coliforms.

The color differential between Escherichia coli colonies and colonies ofother coliforms can be intensified by the incorporation of a contrastpromoter. Without intending to be limited thereby, it is believed thatthe contrast promoters described herein achieve an intensifiedcoloration by increasing the formation of glucuronidase by Escherichiacoli. Promoters particularly suitable for maximizing the formation ofglucuronidase by Escherichia coli are the prime amino acids ofhistidine, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan and valine.

The medium is capable of promoting and sustaining the growth of coliformbacteria to the exclusion of non-coliform bacteria without the inclusionof an anionic or cationic surface active agent. Ionically active surfaceactive agents are commonly employed in membrane filtration media whichinclude a chromogenic substrate, such as a β-glucuronidase, as they openthe bacterial membrane and increase contact between the chromogenicenzymatic substrate and the enzyme produced by the bacteria.Unfortunately, ionically active surface active agents alsoindiscriminately destroy injured bacteria, resulting in a loss of targetbacteria from the inoculated sample. Examples of anionic and cationicsurface active agents frequently used in culture media designed formembrane filtration testing include alkyl sulfates such as sodium laurylsulfate, and bile acids such as deoxycholate and sodium deoxycholate.

A specific formulation found to exhibit the desired bacterialselectivity for coliforms and provide a readily perceiveddifferentiation between Escherichia coli colonies and colonies of othercoliforms, wherein the preferred concentration is listed in brackets "[]", includes:

1. 0.1 to 30 g/l (0.6 g/l) lactose as a carbon nutrient source;

2. 0.01 to 50 g/l (8.0 g/l) of a nitrogen and carbon nutrient containingprotein source, such as casitone;

3. 0.001 to 20 g/l (1.25 g/l) monobasic potassium phosphate and 0.002 to40 g/l (1.75 g/l) dibasic potassium phosphate as metabolizable sourcesof both potassium and phosphate nutrients;

4. 0.01 to 20 (0.5 g/l) yeast extract as a source of vitamins, mineralsand amino acids;

5. 0.01 to 10 g/l (0.3 g/l) anhydrous magnesium sulfate as ametabolizable source of magnesium;

6. 0.01 to 10 g/l (3.0 g/l) sodium chloride as a recovery agent;

7. 0.005 to 40 g/l (1.0 g/l) sodium pyruvate;

8. 0.2 to 10 g/l (0.5 g/l) nonionic surfactant;

9. a bactericidal system selective for non-coliform bacteria of:

(a) 0.0001 to 0.1 g/l (0.015 g/l) methylene blue, and

(b) 0.0001 to 0.05 g/l (0.003 g/l) Erythromycin;

10. 0.001 to 1.0 g/l (0.2 g/l) 5-bromo-4-chloro-3-indolyl-β-D-glucuronicacid cyclohexylammonium salt as a sensible indicator selectivelymetabolized by Escherichia coli to the exclusion of other coliforms;

11. 0.001 to 20 g/l (0.1 g/l) of an amino acid--such as one or more ofthe prime amino acids histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan and valine--as acontrast promoter for increasing the formation of glucuronidase byEscherichia coli; and

12. 0.001 to 0.5 g/l (0.07 g/l) triphenyltetrazolium chloride as ahighlighting dye to enhance visual differentiation of coliform coloniesfrom the culture medium.

The medium may optionally include about 0.8 to 2 g/l agar or othersuitable thickener to gel the medium in accordance with standard gellingtechniques for culture mediums.

The medium is preferably incubated at about 35°±2° C. to encourage thegrowth of coliforms while discouraging growth of various non-coliforms.Detectable results can often be obtained in about 16 hours with anincubation period of 24±4 hours recommended.

Manufacture of Medium

In a first method of manufacture, the heat treatable components of themedium are mixed together in any order to form a first mixture. Thefirst mixture is autoclave sterilized at about 120° C. for 15 minutesand the sterilized first mixture allowed to cool. The heat labilecomponents of the medium are then mixed together in any order, filtersterilized, and combined with the cooled first mixture to form theculture medium.

In a second method of manufacture, all components of the medium aremixed together and filter sterilized.

Regardless of the method used to manufacture the culture medium, therelative concentrations of the various components in themedium--primarily the concentration of monobasic and dibasic potassiumphosphates--are established to provide the medium with a pH of about7±0.2.

Those components which have limited solubility in water, such asβ-glucuronidase 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acidcyclohexylammonium salt and Erythromycin, can be dissolved in a minorproportion of an organic cosolvent, such as ethanol, in order facilitateintroduction of the components into the aqueous based culture medium.The amount of organic cosolvent used should be tightly controlled inorder to limit the concentration of organic cosolvent in the finalculture medium to less than about 3 wt % with a concentration of lessthan about 1 wt % preferred.

The heat treatable components present in the preferred compositioninclude (i) the lactose carbon source, (ii) the casitone nitrogensource, (iii) potassium phosphate, (iv) yeast extract, (v) the magnesiumsource magnesium sulfate, (vi) the nonionic surfactant, (vii) therecovery enhancer sodium chloride, (viii) the bactericidal agentmethylene blue, (ix) the amino acids, and (x) agar.

The heat labile components present in the preferred composition include(i) the recovery enhancer sodium pyruvate, (ii) the bactericidal agenterythromycin, (iii) the chromogenic agent β-glucuronidase5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt,and (iv) the highlighting dye triphenyltetrazolium chloride.

The medium is best suited for use in accordance with the membranefiltration technique but is also suitable for use in accordance withother techniques upon modification of the composition in accordance withstandard changes of such media for use in accordance w/such othertechniques.

The medium is used for test samples such as potentially contaminatedwater and potentially contaminated comestible.

If desired, enumeration of total coliform colonies can be confirmed byconducting a confirmatory test for each of the individual coliformcolonies identified in the culture medium of the present invention. Oneexample of a suitable confirmatory test is the well known oxidase testwhich is described in detail in Standard Methods for the Examination ofWater and Wastewater.

I claim:
 1. A culture medium which provides for selective growth ofcoliforms and simultaneous detection of coliforms and Escherichia colias distinct from other coliforms, and which does so with the use of achromogenic indicator, to the exclusion of use of any fluorogenicindicators, consisting essentially of:a media containing a carbonnutrient source, a nitrogen nutrient source, a source of metabolizablepotassium, a source of metabolizable phosphate, a source of vitamins, asource of minerals, a source of amino acids, and sodium pyruvate; abactericidal system for non-coliform bacteria; a nonionic surfactant forenhancing visibility of bacterial colonies by increasing colonydiameter; a species differentiating chromogenic indicator selected fromthe group consisting of the ammonium and sodium salts of5-bromo-4-chloro-3-indolyl-B-D-glucuronic acid cyclohexane capable ofselective metabolism by Escherichia coli to the exclusion of othercoliforms; and triphenyltetrazolium chloride as a highlighting dyemetabolized by coliforms.
 2. The medium of claim 1 further comprising asource of metabolizable magnesium.
 3. The medium of claim 2 wherein thesource of metabolizable magnesium comprises at least magnesium sulfate.4. The medium of claim 1 further comprising a recovery agent.
 5. Themedium of claim 4 wherein the recovery agent comprises sodium chloride.6. The medium of claim 1 further comprising a contrast promoter forincreasing a color differential between Escherichia coli colonies andcolonies of other coliforms in the medium.
 7. The medium of claim 6wherein the contrast promoter comprises at least one amino acid.
 8. Themedium of claim 7 wherein the amino acid is methionine.
 9. The medium ofclaim 1 further comprising about 0.8 to about 2 g/l solidifying agent.10. The medium of claim 1 wherein the pH of the medium is about 6.8 to7.2.
 11. The medium of claim 1 wherein the carbon nutrient source islactose.
 12. The medium of claim 1 wherein the nitrogen nutrient sourceis casitone.
 13. The medium of claim 1 wherein potassium phosphate isthe source of both potassium and phosphate nutrients.
 14. The medium ofclaim 1 wherein yeast extract is the source of the vitamins, mineralsand amino acids.
 15. The medium of claim 1 wherein the bactericidalsystem is selected from the group consisting of methylene blue,erythromycin and a combination of methylene blue and erythromycin.
 16. Amethod of simultaneously detecting total coliform and Escherichia coliwith a single growth phase incubation period comprising:(a) inoculatingthe culture medium of claim 1 with a potentially coliform contaminatedspecimen, (b) incubating the inoculated medium at about 35°±0.2° C. forabout 16 to 28 hours, and (c) analyzing the incubated medium toseparately identify and quantify the colonies of total coliforms andcolonies of Escherichia coli.
 17. The method of claim 16 wherein thespecimen is potentially contaminated water.
 18. The method of claim 16wherein the specimen is a potentially contaminated comestible.
 19. Themethod of claim 16 wherein the specimen is subjected to membranefiltration prior to being inoculated into the medium.
 20. The method ofclaim 16 wherein the medium has a pH of about 7±0.2.
 21. A selectiveculture medium providing simultaneous detection of total coliforms andEscherichia coli from a single growth phase incubation period consistingessentially of:(a) 0.1 to 30 g/l lactose, (b) 0.01 to 50 g/l peptones,(c) 0.001 to 20 g/l monobasic potassium phosphate, (d) 0.002 to 40 g/ldibasic potassium phosphate, (e) 0.01 to 20 g/l yeast extract, (f) 0.01to 10 g/l magnesium sulfate, (g) 0.01 to 10 g/l sodium chloride, (h)0.005 to 40 g/l sodium pyruvate as a protective agent, (i) 0.2 to 10 g/lnonionic surfactant, (j) a bactericidal system selective fornon-coliform bacteria of:(i) 0.0001 to 0.1 mg/l methylene blue, (ii)0.001 to 0.5 mg/l erythromycin, (k) 0.001 to 1 g/l of an indicatorselectively metabolized by Escherichia coil to the exclusion of othercoliforms selected from the group consisting of the ammonium and sodiumsalts of 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexane; (l)0.1 g/l contrast promoter for enhancing formation of glucuronidase byEscherichia coli growing in the medium; and (m) 0.001 to 0.5 g/ltriphenyltetrazolium chloride as a highlighting dye metabolized bycoliforms;wherein distinct colonies of coliform bacteria grow on themedium.
 22. The medium of claim 21 further comprising about 0.8 to about2 g/l agar.